During development, certain genes are activated to stimulate cell proliferation, survival, migration, invasion, and neovascularization. These genes are often downregulated once organ development is complete. During carcinogenesis, the same genes are often re-activated, stimulating the same properties, but now out of context. Transcribed by one such gene, the homeobox protein and transcription factor Six1 is expressed during embryogenesis where it mediates the proliferation and survival of progenitor cells. It is lost in most differentiated tissues but re-expressed in a number of cancers including breast cancer. We have shown that when Six1 is expressed out of context in adult cells, it aberrantly promotes both proliferation and survival, contributing to tumorigenesis. Our preliminary evidence shows that Six1 also brings about an epithelial-to-mesenchymal-like transition (EMT) in cultured cells and promotes metastasis in vivo. Our proposed experiments are therefore directed at testing the following tri-partite hypothesis: Misexpression of Six1 after development is complete imparts not only a pro-proliferative and survival phenotype, but also a pro-migratory and invasive phenotype, contributing to metastatic disease. Acquisition of the invasive phenotype is due to a Six1-dependent increase in TGF-beta signaling. The resulting aggressive, metastatic mammary tumors remain dependent on Six1 for survival and for their aggressive behavior. In the proposed research we will provide definitive tests of these hypotheses both in vitro and in vivo and begin to decipher the molecular mechanism by which Six1 causes EMT and metastasis, focusing on the TGF-beta signaling pathway. Our aims are: 1) To determine whether TR-I expression and signaling is required for Six1-mediated oncogenic EMT in cultured mammary epithelial cells;2) To determine in xenograft models whether Six1 overexpression is necessary and sufficient to cause metastatic breast disease, through upregulation of TGF-beta signaling;3) To use inducible transgenic models of Six1 overexpression to determine whether the invasive behavior of the resulting mammary tumors is due to TGF-beta signaling, and whether this behavior remains dependent on Six1. Relevance: Six1 is overexpressed in a much higher percentage of metastatic (90%) compared to primary breast cancers (50%) and its overexpression is often correlated with advanced tumor stage and worsened survival. Further, Six1 influences multiple stages of the tumorigenic process. For this reason therapeutic agents targeting Six1 have the potential to inhibit breast cancer at both early and later stages of disease progression. Because Six1 is lost in most adult tissues such therapeutic agents should have limited side effects. Project Narrative: Expression of the Six1 homeobox gene causes both tumor initiation and metastasis. Thus, therapeutic agents targeting Six1 have the potential to inhibit breast cancer at both early and later stages of disease progression. Further, because Six1 is expressed only during embryogenesis, and not in the adult, therapies that target Six1 should inhibit tumor cell growth and metastasis with limited side effects.